本文選題：霍亂弧菌　切入點：上轉(zhuǎn)發(fā)光免疫層析　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：1.研究背景及目的霍亂是由霍亂弧菌引起的一種急性腹瀉性傳染病,其發(fā)病急,傳染性強(qiáng),死亡率高,是《中華人民共和國傳染病防治法》規(guī)定的甲類傳染病之一�，F(xiàn)在霍亂疫情仍然時有報道,嚴(yán)重威脅著人民群眾的健康,快速、簡便、準(zhǔn)確的檢測霍亂尤為重要�，F(xiàn)已證實霍亂弧菌共有超過200種血清群,其中O1群與O139群為主要致病群,其他型別幾乎不會造成疾病流行。因此準(zhǔn)確檢測O1與O139群具有很大的現(xiàn)實意義�，F(xiàn)有的霍亂檢測手段或多或少都存在一些問題,比如有的需要實驗操作人員豐富的經(jīng)驗,有的需要依靠精密的儀器設(shè)備,有的需要嚴(yán)密的實驗設(shè)計,有的容易造成結(jié)果誤判,有的只能定性判斷,有的實驗耗時較長,有的敏感性不是很高,有的實驗成本較高,這些不足都抑制了霍亂弧菌臨床快速檢驗的發(fā)展。2.研究方法免疫層析作為經(jīng)典快速檢驗技術(shù),操作簡便,耗時很短,僅使用制備完成的拭紙,直接將樣品加至樣品墊,靜置一段時間后,即可在視窗肉眼觀察結(jié)果。然而經(jīng)典的免疫層析技術(shù)過于依賴操作者的個人經(jīng)驗,且只能定性判斷結(jié)果,這些缺點都制約了免疫層析的發(fā)展。將上轉(zhuǎn)換發(fā)光材料與免疫層析相結(jié)合,可以很大程度上避免這一問題。上轉(zhuǎn)發(fā)光免疫層析檢測樣品時,沒有雜光干擾,背景干凈,既保留了經(jīng)典免疫層析技術(shù)的優(yōu)點,如操作簡便,設(shè)備輕巧,實驗耗時很少,又解決了經(jīng)典免疫層析的問題,比如結(jié)果判定依賴肉眼觀察,易造成假陽性或假陰性,不能定量判斷等,是一種具有臨床快速檢驗前景的新技術(shù)。本論文通過研究,利用基于上轉(zhuǎn)發(fā)光免疫層析技術(shù)(Up-converting phosphor technology-based immunochromatographic assay,UPT-ICG),采用雙抗體夾心的方法分別制備了3種拭紙——檢測小川型的9B12單抗自配拭紙;檢測霍亂O1的9F11單抗自配拭紙;檢測霍亂主要流行株的M-(9F11+mAb2)+P-(9F11+mAb1);還制備完成了可以檢測不同分辨度霍亂弧菌的三靶標(biāo)拭紙。三靶標(biāo)拭紙可以同時檢測霍亂弧菌O1群小川型、霍亂弧菌O1群與霍亂弧菌O139群,三條檢測帶之間互不干擾,基本可以達(dá)到對霍亂弧菌主要致病群的甄別。通過對拭紙樣品墊成分優(yōu)化、結(jié)合墊抗體量與標(biāo)記條件優(yōu)化、分析膜抗體量優(yōu)化,現(xiàn)在這3種單靶標(biāo)拭紙與1種三靶標(biāo)拭紙可以達(dá)到很好的敏感性、線性、精密性與特異性。經(jīng)過現(xiàn)場評價,4種拭紙均可以達(dá)到預(yù)期實驗結(jié)果,檢測準(zhǔn)確率很高。3.研究結(jié)果我們制備了霍亂弧菌O1群小川型單克隆抗體9B12、霍亂弧菌O1群稻葉型單克隆抗體9F11;并且利用這兩種單克隆抗體,結(jié)合已有的霍亂弧菌O139群抗體mAb1與mAb2制備了三種基于上轉(zhuǎn)發(fā)光免疫層析的單靶標(biāo)拭紙,可以分別檢測霍亂弧菌O1群小川型、霍亂弧菌O1群與霍亂弧菌流行株——即O1群與O139群。其中檢測霍亂弧菌O1群小川型的拭紙M-9B12+P-9B12、檢測霍亂弧菌O1群的拭紙M-9F11+P-9F11的敏感性均為102cfu/ml,檢測霍亂弧菌流行株的拭紙M-(9F11+mAb2)+P-(9F11+mAb1)敏感性為103cfu/ml。檢測其他食源性菌,如沙門氏菌、副溶血弧菌、致病型大腸埃希菌、李斯特菌等時,均為陰性反應(yīng),可見這三種單靶標(biāo)拭紙具有良好的特異性。在中國疾病預(yù)防控制中心進(jìn)行的臨床分離株現(xiàn)場評價實驗,共檢測菌株78株,包括霍亂弧菌O1群小川型15株,稻葉型12株,彥島型12株(與小川型有血清凝集的共5株),O139群15株,非O1非O139群24株。針對霍亂弧菌O1群小川型的M-9B12+P-9B12檢測小川型覆蓋度為100%,檢測各型霍亂弧菌的準(zhǔn)確率為98.7%。針對霍亂弧菌O1群的拭紙M-9F11+P-9F11檢測霍亂O1群的覆蓋度為100%,檢測各型霍亂的準(zhǔn)確率為100%。在三種單靶標(biāo)拭紙的基礎(chǔ)上,我們研發(fā)成功了一種可以同時檢測不同分辨度霍亂弧菌的三靶標(biāo)拭紙,在定性判斷檢測霍亂弧菌樣品的血清群、定量檢測其濃度的基礎(chǔ)上,可以進(jìn)行初步分型。根據(jù)出現(xiàn)峰值的T帶,判定待檢樣品是霍亂弧菌O1群小川型,或是霍亂弧菌O1群,或是霍亂弧菌O139群。這在國內(nèi)外尚屬首創(chuàng)。該三靶標(biāo)拭紙檢測霍亂弧菌的敏感性可達(dá)103cfu/ml,與其他食源性菌,如沙門氏菌、副溶血弧菌、致病型大腸埃希菌、李斯特菌等無交叉反應(yīng),特異性好,在中國疾病預(yù)防控制中心進(jìn)行臨床分離株現(xiàn)場評價實驗,其覆蓋度為100%,檢測各型霍亂的準(zhǔn)確率為100%。我們研發(fā)的4種基于稀土納米上轉(zhuǎn)發(fā)光免疫層析技術(shù)的高靈敏霍亂弧菌檢測拭紙具有操作簡便,儀器易攜帶,實驗耗時短,可以定性定量判定實驗結(jié)果,敏感性與特異性理想等優(yōu)點,是很有潛力的臨床快速檢驗方法。4.討論在現(xiàn)有的霍亂弧菌檢測方法不能完全滿足臨床快速檢驗的要求,存在各種各樣缺陷的情況下,基于稀土納米上轉(zhuǎn)發(fā)光免疫層析技術(shù)的拭紙研發(fā),很大程度上可以彌補(bǔ)這一問題。使用上轉(zhuǎn)發(fā)光免疫層析拭紙檢測菌株時,僅需要使用已配制好的樣品處理液(本論文使用的是堿性蛋白胨水)稀釋樣品,然后使用加樣槍吸取樣品加至拭紙加樣孔內(nèi),靜置15min后使用UPT生物傳感器檢測,即可獲得實驗結(jié)果。一次實驗操作涉及的拭紙、樣品處理液為事前制備完成的;生物傳感器體積輕巧,便于攜帶;實驗操作非常簡單,對實驗操作者沒有任何技術(shù)要求,易于推廣,非常適合臨床快速檢驗及在基層衛(wèi)生醫(yī)療中使用,對霍亂疫情的快速發(fā)現(xiàn)、及時處理、得當(dāng)防控都具有非常重要的意義。
[Abstract]:1. background and objective cholera is caused by Vibrio cholera is an acute infectious disease of diarrhea, the incidence of acute, highly infectious, high mortality, is one of the "infectious diseases prevention and treatment of infectious diseases in People's Republic of China. Now the cholera epidemic > provisions are reported, a serious threat to people's health, fast and convenient accurate detection of cholera, Vibrio cholerae is particularly important. It has been confirmed that there are more than 200 kinds of serum group, O1 group and O139 group as the main pathogenic group, other types can hardly cause epidemic disease. Therefore, accurate detection of O1 and O139 group has great practical significance. Cholera existing detection methods have some problems, such as some experimental operating personnel with rich experience, some need to rely on sophisticated equipment, some require rigorous experimental design, easy to cause the results of some false positives, some can only set The judgment of some experimental time-consuming, some sensitivity is not very high, some experimental research methods.2. development cost is high, these problems are inhibited the rapid detection of Vibrio cholerae clinical immune chromatography as classic rapid detection technology, simple operation, time is very short, only completed with a wipe, directly added to the sample the sample pad, after standing for a long time, can be observed in the naked eye window the results. However immunochromatography classic is too dependent on the experience of the operator, and can only be qualitative judgment, these shortcomings have restricted the development of immunochromatographic. The upconversion luminescence materials and immunochromatography combined, can largely avoid this a problem. To luminescence immunochromatography test samples, no stray light interference, clean background, not only retains the advantages of classical immunochromatographic technique, such as simple operation, light equipment, very time-consuming experiment Less, but also solve the problems such as classical immune chromatography, determination of the naked eyes, easily lead to false positive or false negative, not quantitative judgment, is a new technology with a rapid test clinical prospect. Through the research, the use of transgenic technology based on layer analysis on chemiluminescence immunoassay (Up-converting phosphor technology-based immunochromatographic assay. UPT-ICG), using the method of double antibody sandwich were prepared 3 kinds of wiping paper - Detection Ogawa 9B12 monoclonal antibody with swabs; detection of cholera O1 9F11 monoclonal antibody with swabs; detection of cholera strains M- (9F11+mAb2) +P- (9F11+mAb1); also completed the preparation can detect different resolution the degree of Vibrio cholerae three target wipes. Three target wipes can simultaneously detect Vibrio cholerae O1 serotype Ogawa, Vibrio cholerae O1 and Vibrio cholerae O139 detection, three with no mutual interference between the basic. In order to achieve the main pathogenic Vibrio cholerae group screening. Based on the wiping paper sample pad composition optimization, combination pad antibody quantity and labeling conditions optimization, analysis of membrane antibody quantity optimization, now the 3 single target swabs and 1 kinds of three target wipes can achieve very good sensitivity, linearity, precision and specificity after on-site evaluation, 4 kinds of wiping paper can reach the expected results, the detection accuracy rate is very high.3. our results for monoclonal antibody 9B12 against Vibrio cholerae O1 serotype Ogawa by monoclonal antibody 9F11 against Vibrio cholerae O1 serotype Inaba; and the use of these two kinds of monoclonal antibodies, three based on the turn light immunochromatography single target swabs were prepared with the Vibrio cholerae O139 antibody mAb1 and mAb2 were detected respectively, Vibrio cholerae O1 serotype Ogawa, Vibrio cholerae O1 and Vibrio cholerae strains of O1 group and O139 group. The detection of Cholera Cholerae O1 serotype Ogawa wipe M-9B12+P-9B12, the sensitivity of detection of Vibrio cholerae O1 group M-9F11+P-9F11 swabs were 102cfu/ml for detection of Vibrio cholerae strains were M- (9F11+mAb2) +P- paper (9F11+mAb1) sensitivity for 103cfu/ml. detection of other foodborne pathogens, such as Salmonella, Vibrio parahaemolyticus, pathogenic Escherichia coli, Lester bacteria, were negative, showing that these three single target swabs had good specificity. In clinical isolates of site evaluation experiment China Center for Disease Control and prevention, were detected in 78 strains, including choleraic vibrio O1 serotype 15 strains, 12 strains of rice leaf type, hikojima 12 strains (serum agglutination and Ogawa total 5 strains), 15 strains of group O139, non O1 and non O139 group. 24 strains of M-9B12+P-9B12 detection for Vibrio cholerae Ogawa serotype Ogawa O1 coverage is 100%, the detection rate of each type of Vibrio cholerae 98.7%. in Huo Chaos wipes M-9F11+P-9F11 group O1 detection of Vibrio cholera O1 group coverage is 100%, the detection rate of each type of cholera 100%. based on three kinds of single target wipes on, we successfully developed a can simultaneously detect different resolution of Vibrio cholerae three target swabs, determine the detection of Vibrio cholerae samples in the qualitative and quantitative detection of serum group, based on its concentration, can make a preliminary classification. According to the peak value of T, to determine the sample to be detected is Vibrio cholerae O1 serotype Ogawa, and Vibrio cholerae O1, or Vibrio cholerae O139. This is the first at home and abroad. The three target samples paper detection of Vibrio Cholera sensitivity up to 103cfu/ml, and other food borne bacteria, such as Salmonella, Vibrio parahaemolyticus, pathogenic Escherichia coli and other bacteria, no cross reaction to Lester, good specificity, in Chinese disease prevention and control center for clinical isolates of the scene The experimental evaluation and its coverage is 100%, the detection rate of various types of cholera 100%. we developed 4 kinds of luminescence immunochromatography technology based on rare earth nano on high sensitive detection of Vibrio cholerae wipes instrument has the advantages of simple operation, easy to carry, the short time, can determine the qualitative and quantitative experimental results, the sensitivity and specificity of ideal the advantages of.4. clinical rapid test methods have the potential to discuss completely meet the clinical requirements in rapid detection of Vibrio cholerae not existing detection methods, there are various kinds of defects in the case of R & d wipe paper converting phosphor immunochromatography nano based on great extent can make up for this problem. Chemiluminescence immunoassay the use of paper chromatography swab was detected, only need to use liquid sample has been prepared (this paper is using alkaline peptone water) dilution of the sample, and then use the sample suction gun The sample is added to the wiping paper sample adding hole, static 15min after using UPT biosensor, obtained experimental results. An experimental operation involving a wipe, liquid sample preparation prior to complete system; biosensor is lightweight, easy to carry; the operation is very simple, no technical requirements for the operator is easy to popularize, very suitable for clinical use and rapid test in the primary health care, to quickly find the cholera epidemic, timely treatment, has a very important significance for prevention and control properly.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R516.5;R446.6

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